Nef-induced STAT1 activation in MDMs is mediated by soluble factor release.

(A) Western blot analysis of phosphotyrosine-STAT1, STAT1, or β-tubulin levels performed on total cell extracts of MDMs treated for 30 and 60 minutes with supernatants collected from MDMs of the same donors treated with 100 ng/mL Nef for 2 hours after immunodepletion for the presence of residual rNef. Cell extracts from MDMs treated with supernatants from untreated MDMs (Ctrl) or directly treated for 2 hours with 100 ng/mL rNef served as controls. Cell extracts analyzed (A,B) were obtained by pooling simultaneous cell cultures from 4 healthy donors each. (B) Western blot analysis of phosphotyrosine-STAT1, STAT1, or β-tubulin in MDMs treated with supernatants collected from MDMs infected with VSV-G pseudotyped HIV-1 expressing the full length (fl) or defective HIV-1 genomes. Eight hours after infection, supernatants were clarified from residual viral particles through ultracentrifugation and added to fresh MDMs of the same donors. After an incubation period of 60 minutes, cells were collected and total cell extracts assayed. Cell extracts from MDMs treated with supernatants from uninfected cells (Ctrl) or incubated for 2 hours with 100 ng/mL rNef were used as controls. Analyzed cell extracts were obtained by pooling simultaneous cell cultures from 3 healthy donors. In both panels, specific signals are indicated on the left side; molecular size markers (in kilodaltons) are reported on the right.